GSM6767514: YUMM1.7 cells, cell bodies, biol rep 2; Mus musculus; RNA-Seq
PET Millicell Hanging Cell Culture Inserts with 1 mm pores sized for 6 well plates were coated on the bottom side with 30 ug/mL fibronectin. Fibronectin was diluted in sterile PBS and inserts were coated for 15 minutes, then aspirated and allowed to dry. 6x105 to 8x105 cells were plated per membrane and placed in an incubator overnight. For fractionation, media was aspirated, cells were rinsed with RNase-free PBS and aspirated, then cell bodies were scraped using a cell scraper and collected into Qiazol. Inserts were rinsed with RNase-free PBS and cleaned with a cotton swab three times. The cleaned insert was carefully dislodged, placed inside a new tube with Qiazol and briefly vortexed. After cell bodies and protrusions were collected, the standard protocol the Qiagen miRNeasy kit was followed. RNA was eluted with 30 μl RNase-free water. There were two RNA seq replicates, each with 6 inserts. 0.69 - 9.4 ug Total RNA was extracted for each sample. Samples are run on the Agilent Tapestation 4200 to determine level of degradation thus ensuring  only high quality RNA is used (RIN Score 8 or higher).   The Qubit fluorometer is used to determine the concentration prior to staring library prep.  One microgram of total DNAse treated RNA is then prepared with the TruSeq Stranded Total RNA LT Sample Prep Kit from Illumina. Total RNA is depleted of its rRNA and fragmented before strand specific cDNA synthesis. cDNA are then a-tailed and indexed adapters are ligated.   After adapter ligation, samples are PCR amplified and purified with AmpureXP beads, then validated again on the Agilent Tapestation 4200.  Before being normalized and pooled, samples are quantified by Qubit then run on the Illumina NextSeq 500 using V2.5 reagents.